在線固相萃取—超高效液相色譜—串聯(lián)質(zhì)譜法

張品等

摘 要 建立了乳制品中三氯生、三氯卡班、雙酚A和壬基酚4種內(nèi)分泌干擾物的在線固相萃取超高壓液相色譜串聯(lián)質(zhì)譜（Online SPE LCMS/MS）檢測方法。液態(tài)乳制品或奶粉樣品中加入乙酸緩沖液，目標物經(jīng)β葡糖醛酸苷肽酶/芳基磺酸酯酶酶解后， 用乙腈提取， 冷凍離心10 min后，取上清液，用水稀釋，在線固相萃取串聯(lián)質(zhì)譜法測定。樣品溶液經(jīng)Xbridge C8柱富集，BEH C18色譜柱分離，甲醇和水梯度洗脫，三重四極桿質(zhì)譜電噴霧負離子模式下采集數(shù)據(jù)，同位素內(nèi)標法定量。4種目標化合物的線性范圍為0.005～5.0 μg/L，相關(guān)系數(shù)R2>0.99；方法的定量限為0.03～1.0 μg/kg，3個添加水平的平均加標回收率為80.2%～106.7%，

3.2 提取條件的優(yōu)化

環(huán)境污染物或藥物進入生物體后在肝臟酶系統(tǒng)的作用下會轉(zhuǎn)化成葡糖糖醛酸結(jié)合態(tài)。Zhou等[ 18]發(fā)現(xiàn)人體尿液中的三氯卡班及其代謝產(chǎn)物主要以結(jié)合態(tài)形式存在，盡管奶樣中是否存在結(jié)合態(tài)還未有定論[ 19，20]，但為了保證檢測結(jié)果的準確性，本研究采用β葡糖醛酸苷肽酶/芳基磺酸酯酶進行酶解。

由于4種目標化合物均有一定的親脂性，因此，酶解后的樣品提取過程比較了乙腈和甲醇兩種與水互溶的極性有機溶劑。稱取適量液奶樣品，加標量為5 μg/kg，如2.3節(jié)所述進行處理，每種提取溶劑做3個平行。離心后提取液直接測定，從表觀上看，乙腈提取液比甲醇提取液更清澈透明。結(jié)果表明，乙腈提取液對TCS，TCC，NP和BPA的絕對回收率分別為95%，89%，91%和78%，比甲醇體系略高（分別為90%，85%，92%和69%）。綜合表觀結(jié)果和回收率數(shù)據(jù)，確定乙腈為提取溶劑。

3.3 背景污染的考察

由于TCS，NP和BPA廣泛應(yīng)用于多個領(lǐng)域，因此這幾種污染物在環(huán)境中普遍存在，甚至在Milpore的超純水中檢測到了BPA[ 21]。而且，塑料器皿或橡膠制品等都會遷移出大量NP 和BPA，這些均會對分析結(jié)果造成干擾。由于離線固相萃取小柱的篩板為聚丙烯等塑料材質(zhì)，有機溶劑浸潤時會持續(xù)溶出NP，其濃度大于0.2 μg/L[ 17]；使用二氯甲烷、正己烷等非極性有機溶劑進行液液萃取時，其背景污染的水平視溶劑體積、純度、批號及濃縮方式有所不同，以10 mL的農(nóng)殘級二氯甲烷為例（揮干后1 mL甲醇定容測定），旋轉(zhuǎn)蒸發(fā)至干時NP的背景污染水平約為0.15 μg/L，若使用氮氣吹干，背景污染水平則高達1.0 μg/L。

本實驗所用實驗器具均為玻璃材質(zhì)，液相色譜管路為聚四氟乙烯，在線固相萃取柱為不銹鋼柱，所有溶劑均為LCMS級。過程空白中TCC的背景污染水平低于0.005 μg/L，其余3種物質(zhì)TCS、NP和BPA均低于0.05 μg/L，且較穩(wěn)定。該結(jié)果明顯低于離線固相萃取、液液萃取等方法所致的背景污染。究其原因，本方法前處理方法步驟簡單，可引入污染的環(huán)節(jié)少；溶劑耗費量低，減少了實驗試劑可能的污染；本方法的自動化程度高，使得背景污染穩(wěn)定性高。

3.4 線性范圍和定量限

Fig.1 LCMS chromatograms of the target compounds as well as the internal standards in different samples （A： procedural blank sample； B： standard solution， in which the concentration of TCC is 0.05 μg/L， while BPA， NP and TCS are 0.5 μg/L； C： a positive sample， pure milk）[HT5][TS）]

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Determination of 4 Environmental Endocrine

Disruptors Involving Bisphenol A in Dairy Products

by Online Solid Phase Extraction Coupled with

Liquid ChromatographyTandem Mass Spectrometry

ZHANG Pin1，2， ZHANG Jing1，2， CHEN JieJun3， DUAN HeJun2， SHAO Bing*1，2

1（School of Public Health， Capital Medical University， Beijing 100058， China）

2（Beijing Key Laboratory of Diagrostic and Traceability Technologies Food Poisoning，

Beijing Centers for Preventive Medical Research， Beijing 100013， China）

3（China National Center for Biotechnology Development， Beijing 100038， China）

Abstract A simple analytical method by means of online solid phase extraction followed liquid chromatographytandem mass spectrometry （SPELCMS/MS） was developed for the simultaneous quantitation of 4 endocrine disruptors （triclosan， triclocarban， bisphenol A and nonylphenol） in dairy products. Infant formula and milk samples were dissolved in acetic acid buffer and hydrolyzed by βglucuronidase/arylsulfatase. Acetonitrile was used as the extract. Then， the mixture was freezecentrifuged for 10 min and the supernatant was diluted with water， and analyzed via online SPELCMS/MS. The sample extracts were concentrated by an Xbridge C8 cartridge and separated on a BEH C18 column with a gradient mobile phase of methanol and water； then analyzed by triple quadrupole mass spectrometry. Mass acquisition was conducted under negative electrospray ionization mode. Quantification was performed by isotopic internal standard calibration. Acceptable linearity （R2>0.99） was achieved over the range of 0.005-5.0 μg/L， with limits of quantification of 0.03-1.0 μg/kg. Average recoveries of four target compounds （spiked at three concentration levels） ranged from 80.2%-106.7%，with relative standard deviation less than 15%. Due to its rapidity， simplicity， and high sensitivity， the method is suitable for the analysis of endocrine disruptors in dairy products. It has been applied in the analysis of raw milk and milk products collected in Beijing. As a result， nonylphenol was found with a high detectable frequency.

Keywords Dairy products； Triclosan； Bisphenol A； Nonylphenol； Online solid phase extraction liquid chromatographytandem mass spectrometry

（Received 24 September 2014； accepted 7 October 2014）

This work was supported by the National Natural Science Foundation of China （No.21177014）

2（Beijing Key Laboratory of Diagrostic and Traceability Technologies Food Poisoning，

Beijing Centers for Preventive Medical Research， Beijing 100013， China）

3（China National Center for Biotechnology Development， Beijing 100038， China）

Abstract A simple analytical method by means of online solid phase extraction followed liquid chromatographytandem mass spectrometry （SPELCMS/MS） was developed for the simultaneous quantitation of 4 endocrine disruptors （triclosan， triclocarban， bisphenol A and nonylphenol） in dairy products. Infant formula and milk samples were dissolved in acetic acid buffer and hydrolyzed by βglucuronidase/arylsulfatase. Acetonitrile was used as the extract. Then， the mixture was freezecentrifuged for 10 min and the supernatant was diluted with water， and analyzed via online SPELCMS/MS. The sample extracts were concentrated by an Xbridge C8 cartridge and separated on a BEH C18 column with a gradient mobile phase of methanol and water； then analyzed by triple quadrupole mass spectrometry. Mass acquisition was conducted under negative electrospray ionization mode. Quantification was performed by isotopic internal standard calibration. Acceptable linearity （R2>0.99） was achieved over the range of 0.005-5.0 μg/L， with limits of quantification of 0.03-1.0 μg/kg. Average recoveries of four target compounds （spiked at three concentration levels） ranged from 80.2%-106.7%，with relative standard deviation less than 15%. Due to its rapidity， simplicity， and high sensitivity， the method is suitable for the analysis of endocrine disruptors in dairy products. It has been applied in the analysis of raw milk and milk products collected in Beijing. As a result， nonylphenol was found with a high detectable frequency.

Keywords Dairy products； Triclosan； Bisphenol A； Nonylphenol； Online solid phase extraction liquid chromatographytandem mass spectrometry

（Received 24 September 2014； accepted 7 October 2014）

This work was supported by the National Natural Science Foundation of China （No.21177014）

2（Beijing Key Laboratory of Diagrostic and Traceability Technologies Food Poisoning，

Beijing Centers for Preventive Medical Research， Beijing 100013， China）

3（China National Center for Biotechnology Development， Beijing 100038， China）

Abstract A simple analytical method by means of online solid phase extraction followed liquid chromatographytandem mass spectrometry （SPELCMS/MS） was developed for the simultaneous quantitation of 4 endocrine disruptors （triclosan， triclocarban， bisphenol A and nonylphenol） in dairy products. Infant formula and milk samples were dissolved in acetic acid buffer and hydrolyzed by βglucuronidase/arylsulfatase. Acetonitrile was used as the extract. Then， the mixture was freezecentrifuged for 10 min and the supernatant was diluted with water， and analyzed via online SPELCMS/MS. The sample extracts were concentrated by an Xbridge C8 cartridge and separated on a BEH C18 column with a gradient mobile phase of methanol and water； then analyzed by triple quadrupole mass spectrometry. Mass acquisition was conducted under negative electrospray ionization mode. Quantification was performed by isotopic internal standard calibration. Acceptable linearity （R2>0.99） was achieved over the range of 0.005-5.0 μg/L， with limits of quantification of 0.03-1.0 μg/kg. Average recoveries of four target compounds （spiked at three concentration levels） ranged from 80.2%-106.7%，with relative standard deviation less than 15%. Due to its rapidity， simplicity， and high sensitivity， the method is suitable for the analysis of endocrine disruptors in dairy products. It has been applied in the analysis of raw milk and milk products collected in Beijing. As a result， nonylphenol was found with a high detectable frequency.

Keywords Dairy products； Triclosan； Bisphenol A； Nonylphenol； Online solid phase extraction liquid chromatographytandem mass spectrometry

（Received 24 September 2014； accepted 7 October 2014）

This work was supported by the National Natural Science Foundation of China （No.21177014）